Publications by authors named "Sebastian Attig"

20 Publications

  • Page 1 of 1

Gating Harmonization Guidelines for Intracellular Cytokine Staining Validated in Second International Multiconsortia Proficiency Panel Conducted by Cancer Immunotherapy Consortium (CIC/CRI).

Cytometry A 2021 Jan 2;99(1):107-116. Epub 2020 Nov 2.

Elicio Therapeutics, Cambridge, Massachusetts, 02139, USA.

Results from the first gating proficiency panel of intracellular cytokine staining (ICS) highlighted the value of using a consensus gating approach to reduce the variability across laboratories in reported %CD8 or %CD4 cytokine-positive cells. Based on the data analysis from the first proficiency panel, harmonization guidelines for a consensus gating protocol were proposed. To validate the recommendations from the first panel and to examine factors that were not included in the first panel, a second ICS gating proficiency panel was organized. All participants analyzed the same set of Flow Cytometry Standard (FCS) files using their own gating protocol. An optional learning module was provided to demonstrate how to apply the previously established gating recommendations and harmonization guidelines to actual ICS data files. Eighty-three participants took part in this proficiency panel. The results from this proficiency panel confirmed the harmonization guidelines from the first panel. These recommendations addressed the (1) placement of the cytokine-positive gate, (2) identification of CD4 CD8 double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and (6) proper adjustment of the biexponential scaling. In addition, based on the results of this proficiency gating panel, two new recommendations were added to expand the harmonization guidelines: (1) inclusion of dump channel marker to gate all live and dump negative cells and (2) backgating to confirm the correct placement of gates across all populations. © 2020 International Society for Advancement of Cytometry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.24244DOI Listing
January 2021

An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma.

Nature 2020 09 29;585(7823):107-112. Epub 2020 Jul 29.

BioNTech, Mainz, Germany.

Treating patients who have cancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with treatment-refractory tumours. We are testing melanoma FixVac (BNT111)-an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma-in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4 and CD8 T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2537-9DOI Listing
September 2020

Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer.

Nature 2017 07 5;547(7662):222-226. Epub 2017 Jul 5.

CI3 - Cluster for Individualized Immunointervention e.V, Hölderlinstraße 8, 55131 Mainz, Germany.

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature23003DOI Listing
July 2017

CD19 Isoforms Enabling Resistance to CART-19 Immunotherapy Are Expressed in B-ALL Patients at Initial Diagnosis.

J Immunother 2017 06;40(5):187-195

*Section of Pediatric Oncology, Children's Hospital, University Medical Center of the Johannes Gutenberg University, Mainz, Germany ‡Third Department of Medicine, University Medical Center of the Johannes Gutenberg University §Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University †University Cancer Center of the Johannes Gutenberg University, Mainz, Germany.

B-cell acute lymphoblastic leukemia (B-ALL) is the commonest childhood cancer and the prognosis of children with relapsed or therapy refractory disease remains a challenge. Treatment with chimeric antigen receptor-modified T cells targeting the CD19 antigen (CART-19 therapy) has been presented as a promising approach toward improving the outcome of relapsed or refractory disease. However, 10%-20% of the patients suffer another relapse. Epitope-loss under therapy pressure has been suggested as a mechanism of tumor cells to escape the recognition from CART-19 therapy. In this work, we analyzed the expression of CD19 isoforms in a cohort of 14 children with CD19 B-ALL and 6 nonleukemia donors. We showed that an alternatively spliced CD19 mRNA isoform lacking exon 2, and therefore the CART-19 epitope, but not isoforms lacking the transmembrane and cytosolic domains are expressed in leukemic blasts at diagnosis in children and in the bone marrow of nonleukemia donors. Furthermore, we clarified the sequence of a further isoform lacking the epitope recognized by CART-19 therapy and disclosed the presence of new isoforms. In comparison with the children, we showed that alternatively spliced CD19 mRNA isoforms affecting exon 2 are also expressed in 6 adult patients with CD19 B-ALL. On top of that, one of the adults expressed an isoform lacking the CD19 transmembrane and cytosolic domains. In conclusion, we proved that some of the CD19 isoforms contributing to CART-19 escape already preexist at diagnosis and could evolve as a dominant clone during CART-19 therapy suggesting the application of combined treatment approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/CJI.0000000000000169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424577PMC
June 2017

Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy.

Nature 2016 06 1;534(7607):396-401. Epub 2016 Jun 1.

TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University gGmbH, Freiligrathstr. 12, Mainz 55131, Germany.

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature18300DOI Listing
June 2016

Generation of TCR-engineered T cells and their use to control the performance of T cell assays.

J Immunol 2015 Jun 8;194(12):6177-89. Epub 2015 May 8.

Translational Oncology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany;

The systematic assessment of the human immune system bears huge potential to guide rational development of novel immunotherapies and clinical decision making. Multiple assays to monitor the quantity, phenotype, and function of Ag-specific T cells are commonly used to unravel patients' immune signatures in various disease settings and during therapeutic interventions. When compared with tests measuring soluble analytes, cellular immune assays have a higher variation, which is a major technical factor limiting their broad adoption in clinical immunology. The key solution may arise from continuous control of assay performance using TCR-engineered reference samples. We developed a simple, stable, robust, and scalable technology to generate reference samples that contain defined numbers of functional Ag-specific T cells. First, we show that RNA-engineered lymphocytes, equipped with selected TCRs, can repetitively deliver functional readouts of a controlled size across multiple assay platforms. We further describe a concept for the application of TCR-engineered reference samples to keep assay performance within or across institutions under tight control. Finally, we provide evidence that these novel control reagents can sensitively detect assay variation resulting from typical sources of error, such as low cell quality, loss of reagent stability, suboptimal hardware settings, or inaccurate gating.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1400958DOI Listing
June 2015

Data analysis as a source of variability of the HLA-peptide multimer assay: from manual gating to automated recognition of cell clusters.

Cancer Immunol Immunother 2015 May 18;64(5):585-98. Epub 2015 Feb 18.

Department of Immunology, Institute for Cell Biology, Eberhard Karls University, Auf der Morgenstelle 15, 72076, Tübingen, Germany,

Multiparameter flow cytometry is an indispensable method for assessing antigen-specific T cells in basic research and cancer immunotherapy. Proficiency panels have shown that cell sample processing, test protocols and data analysis may all contribute to the variability of the results obtained by laboratories performing ex vivo T cell immune monitoring. In particular, analysis currently relies on a manual, step-by-step strategy employing serial gating decisions based on visual inspection of one- or two-dimensional plots. It is therefore operator dependent and subjective. In the context of continuing efforts to support inter-laboratory T cell assay harmonization, the CIMT Immunoguiding Program organized its third proficiency panel dedicated to the detection of antigen-specific CD8(+) T cells by HLA-peptide multimer staining. We first assessed the contribution of manual data analysis to the variability of reported T cell frequencies within a group of laboratories staining and analyzing the same cell samples with their own reagents and protocols. The results show that data analysis is a source of variation in the multimer assay outcome. To evaluate whether an automated analysis approach can reduce variability of proficiency panel data, we used a hierarchical statistical mixture model to identify cell clusters. Challenges for automated analysis were the need to process non-standardized data sets from multiple centers, and the fact that the antigen-specific cell frequencies were very low in most samples. We show that this automated method can circumvent difficulties inherent to manual gating strategies and is broadly applicable for experiments performed with heterogeneous protocols and reagents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00262-014-1649-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528367PMC
May 2015

Functional TCR retrieval from single antigen-specific human T cells reveals multiple novel epitopes.

Cancer Immunol Res 2014 Dec 22;2(12):1230-44. Epub 2014 Sep 22.

Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany. Translational Oncology at the University Medical Center, Johannes Gutenberg University, Mainz gGmbH, Germany.

The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-specific CD8(+) or CD4(+) T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8(+) and CD4(+) T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2326-6066.CIR-14-0108DOI Listing
December 2014

Phenotyping of peripheral blood mononuclear cells of patients with advanced heavily pre-treated adenocarcinoma of the stomach and gastro-esophageal junction.

Cancer Immunol Immunother 2014 Dec 28;63(12):1273-84. Epub 2014 Aug 28.

Ganymed Pharmaceuticals AG, An der Goldgrube 12, 55131, Mainz, Germany.

Immunotherapeutic approaches are emerging as promising new treatment options for patients with solid cancers. The host immune system in cancer patients is dysfunctional due to a number of reasons. The level of immunosuppression is variable at the time of diagnosis and depends on the particular cancer entity, stage, and prior anti-cancer therapies. For many cancer entities, the immune alterations of the respective patient population have not been further characterized even though a patient's immunophenotype may be prognostic for the course of the disease or predictive for clinical/biological response to immunotherapy. In this study, we used flow cytometry to determine the phenotype of peripheral blood mononuclear cells (PBMCs) from 30 patients with heavily pre-treated, advanced adenocarcinoma of the stomach and gastro-esophageal junction. The frequencies and activation status of relevant immune effector populations were determined in PBMCs and compared to those of healthy individuals. This report provides comprehensive immune phenotyping data of a patient population with a high medical need.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00262-014-1596-xDOI Listing
December 2014

mTOR inhibition improves antitumor effects of vaccination with antigen-encoding RNA.

Cancer Immunol Res 2013 Dec 20;1(6):386-92. Epub 2013 Sep 20.

Authors' Affiliations: Ganymed Pharmaceuticals, Mainz, Germany.

Vaccination with in vitro transcribed RNA encoding tumor antigens is an emerging approach in cancer immunotherapy. Attempting to further improve RNA vaccine efficacy, we have explored combining RNA with immunomodulators such as rapamycin. Rapamycin, the inhibitor of mTOR, was used originally for immunosuppression. Recent reports in mouse systems, however, suggest that mTOR inhibition may enhance the formation and differentiation of the memory CD8(+) T-cell pool. Because memory T-cell formation is critical to the outcome of vaccination approaches, we studied the impact of rapamycin on the in vivo primed RNA vaccine-induced immune response using the chicken ovalbumin-expressing B16 melanoma model in C57BL/6 mice. Our data show that treatment with rapamycin at the effector-to-memory transition phase skews the vaccine-induced immune response toward the formation of a quantitatively and qualitatively superior memory pool and results in a better recall response. Tumor-infiltrating immune cells from these mice display a favorable ratio of effector versus suppressor cell populations. Survival of mice treated with the combined regimen of RNA vaccination with rapamycin is significantly longer (91.5 days) than that in the control groups receiving only one of these compounds (32 and 46 days, respectively). Our findings indicate that rapamycin enhances therapeutic efficacy of antigen-specific CD8(+) T cells induced by RNA vaccination, and we propose further clinical exploration of rapamycin as a component of immunotherapeutic regimens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2326-6066.CIR-13-0046DOI Listing
December 2013

Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins.

Cytometry A 2014 Jul 19;85(7):621-7. Epub 2014 Feb 19.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz, 55131, Mainz, Germany.

Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.22451DOI Listing
July 2014

CIMT 2013: advancing targeted therapies--report on the 11th Annual Meeting of the Association for Cancer Immunotherapy, May 14-16 2013, Mainz, Germany.

Hum Vaccin Immunother 2013 Sep 22;9(9):2025-32. Epub 2013 Jul 22.

TRON-Translational Oncology at the University Medical Center of Johannes Gutenberg University; Mainz, Germany.

The 11th Annual Meeting of Association for Cancer Immunotherapy (CIMT) welcomed more than 700 scientists around the world to Mainz, Germany and continued to be the largest immunotherapy meeting in Europe. Renowned speakers from various fields of cancer immunotherapy gave lectures under CIMT2013's tag: "Advancing targeted therapies" the highlights of which are summarized in this meeting report.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/hv.25768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906376PMC
September 2013

Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols.

Cancer Immunol Immunother 2013 Apr 9;62(4):615-27. Epub 2012 Nov 9.

III. Medical Department, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.

Robust and sensitive ELISPOT protocols are commonly applied concomitant with the development of new immunotherapeutics. Despite the knowledge that individual serum batches differ in their composition and may change properties over time, serum is still commonly used in immunologic assays. Commercially available serum batches are expensive, limited in quantity and need to be pretested for suitability in immunologic assays, which is a laborious process. The aim of this study was to test whether serum-free freezing media can lead to high cell viability and favorable performance across multiple ELISPOT assay protocols. Thirty-one laboratories from ten countries participated in a proficiency panel organized by the Cancer Immunotherapy Immunoguiding Program to test the influence of different freezing media on cell quality and immunologic function. Each center received peripheral blood mononuclear cells which were frozen in three different media. The participants were asked to quantify antigen-specific CD8+ T-cell responses against model antigens using their locally established IFN-gamma ELISPOT protocols. Self-made and commercially available serum-free freezing media led to higher cell viability and similar cell recovery after thawing and resting compared to freezing media supplemented with human serum. Furthermore, the test performance as determined by (1) background spot production, (2) replicate variation, (3) frequency of detected antigen-specific spots and (4) response detection rate was similar for serum and serum-free conditions. We conclude that defined and accessible serum-free freezing media should be recommended for freezing cells stored for subsequent ELISPOT analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00262-012-1359-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624011PMC
April 2013

Promiscuous survivin peptide induces robust CD4+ T-cell responses in the majority of vaccinated cancer patients.

Int J Cancer 2012 Jul 14;131(1):140-9. Epub 2011 Sep 14.

Department of Immunology, Institute for Cell Biology, Eberhard Karls University, Tübingen 72076, Germany.

CD4(+) T cells have been shown to be crucial for the induction and maintenance of cytotoxic T cell responses and to be also capable of mediating direct tumor rejection. Therefore, the anticancer therapeutic efficacy of peptide-based vaccines may be improved by addition of HLA class II epitopes to stimulate T helper cells. Survivin is an apoptosis inhibiting protein frequently overexpressed in tumors. Here we describe the first immunological evaluation of a survivin-derived CD4(+) T cell epitope in a multipeptide immunotherapy trial for prostate carcinoma patients. The survivin peptide is promiscuously presented by several human HLA-DRB1 molecules and, most importantly, is naturally processed by dendritic cells. In vaccinated patients, it was able to induce frequent, robust and multifunctional CD4(+) T cell responses, as monitored by IFN-γ ELISPOT and intracellular cytokine staining. Thus, this HLA-DR restricted epitope is broadly immunogenic and should be valuable for stimulating T helper cells in patients suffering from a wide range of tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ijc.26365DOI Listing
July 2012

FLT3 ligand enhances the cancer therapeutic potency of naked RNA vaccines.

Cancer Res 2011 Oct 4;71(19):6132-42. Epub 2011 Aug 4.

Institute for Translational Oncology and Immunology, Mainz, Germany.

Intranodal immunization with antigen-encoding naked RNA may offer a simple and safe approach to induce antitumor immunity. RNA taken up by nodal dendritic cells (DC) coactivates toll-like receptor (TLR) signaling that will prime and expand antigen-specific T cells. In this study, we show that RNA vaccination can be optimized by coadministration of the DC-activating Fms-like tyrosine kinase 3 (FLT3) ligand as an effective adjuvant. Systemic administration of FLT3 ligand prior to immunization enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodal RNA. Unexpectedly, plasmacytoid DCs (pDC) were found to be essential for the adjuvant effect of FLT3 ligand and they were systemically expanded together with conventional DCs after treatment. In response to FLT3 ligand, pDCs maintained an immature phenotype, internalized RNA, and presented the RNA-encoded antigen for efficient induction of antigen-specific CD8(+) T-cell responses. Coadministration of FLT3 ligand with RNA vaccination achieved remarkable cure rates and survival of mice with advanced melanoma. Our findings show how to improve the simple and safe strategy offered by RNA vaccines for cancer immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-11-0291DOI Listing
October 2011

A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols.

J Transl Med 2011 Jul 11;9:108. Epub 2011 Jul 11.

Division of Translational and Experimental Oncology, Department of Internal Medicine III, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.

Background: The introduction of antibody markers to identify undesired cell populations in flow-cytometry based assays, so called DUMP channel markers, has become a practice in an increasing number of labs performing HLA-peptide multimer assays. However, the impact of the introduction of a DUMP channel in multimer assays has so far not been systematically investigated across a broad variety of protocols.

Methods: The Cancer Research Institute's Cancer Immunotherapy Consortium (CRI-CIC) conducted a multimer proficiency panel with a specific focus on the impact of DUMP channel use. The panel design allowed individual laboratories to use their own protocol for thawing, staining, gating, and data analysis. Each experiment was performed twice and in parallel, with and without the application of a dump channel strategy.

Results: The introduction of a DUMP channel is an effective measure to reduce the amount of non-specific MULTIMER binding to T cells. Beneficial effects for the use of a DUMP channel were observed across a wide range of individual laboratories and for all tested donor-antigen combinations. In 48% of experiments we observed a reduction of the background MULTIMER-binding. In this subgroup of experiments the median background reduction observed after introduction of a DUMP channel was 0.053%.

Conclusions: We conclude that appropriate use of a DUMP channel can significantly reduce background staining across a large fraction of protocols and improve the ability to accurately detect and quantify the frequency of antigen-specific T cells by multimer reagents. Thus, use of a DUMP channel may become crucial for detecting low frequency antigen-specific immune responses. Further recommendations on assay performance and data presentation guidelines for publication of MULTIMER experimental data are provided.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1479-5876-9-108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3148571PMC
July 2011

Simultaneous infiltration of polyfunctional effector and suppressor T cells into renal cell carcinomas.

Cancer Res 2009 Nov 20;69(21):8412-9. Epub 2009 Oct 20.

Department of Immunology, Institute for Cell Biology, Eberhard-Karls University, Tübingen, Germany.

Renal cell carcinoma is frequently infiltrated by cells of the immune system. This makes it important to understand interactions between cancer cells and immune cells so they can be manipulated to bring clinical benefit. Here, we analyze subsets and functions of T lymphocytes infiltrating renal cell tumors directly ex vivo following mechanical disaggregation and without any culture step. Subpopulations of memory and effector CD4(+) Th1, Th2, and Th17 and CD8(+) Tc1 cells were identified based on surface phenotype, activation potential, and multicytokine production. Compared with the same patient's peripheral blood, T lymphocytes present inside tumors were found to be enriched in functional CD4(+) cells of the Th1 lineage and in effector memory CD8(+) cells. Additionally, several populations of CD4(+) and CD8(+) regulatory T cells were identified that may synergize to locally dampen antitumor T-cell responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-09-0852DOI Listing
November 2009

Cytomegalovirus infection: a driving force in human T cell immunosenescence.

Ann N Y Acad Sci 2007 Oct;1114:23-35

Department of Immunology, University of Tübingen, Auf der Morgenstelle 15, D-72076, Tübingen, Germany.

The human immune system evolved to defend the organism against pathogens, but is clearly less well able to do so in the elderly, resulting in greater morbidity and mortality due to infectious disease in old people, and higher healthcare costs. Many age-associated immune alterations have been reported over the years, of which probably the changes in T cell immunity, often manifested dramatically as large clonal expansions of cells of limited antigen specificity together with a marked shrinkage of the T cell antigen receptor repertoire, are the most notable. It has recently emerged that the common herpesvirus, cytomegalovirus (CMV), which establishes persistent, life-long infection, usually asymptomatically, may well be the driving force behind clonal expansions and altered phenotypes and functions of CD8 cells seen in most old people. In those few who are not CMV-infected, another even more common herpesvirus, the Epstein-Barr virus, appears to have the same effect. These virus-driven changes are less marked in "successfully aged" centenarians, but most marked in people whom longitudinal studies have shown to be at higher risk of death, that is, those possessing an "immune risk profile" (IRP) characterized by an inverted CD4:8 ratio (caused by the accumulation primarily of CD8(+) CD28(-) cells). These findings support the hypothesis that persistent herpesviruses, especially CMV, act as chronic antigenic stressors and play a major causative role in immunosenescence and associated mortality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1196/annals.1396.043DOI Listing
October 2007

Report on the Fifth Annual Meeting of the Association for Immunotherapy of Cancer (CIMT) April 12-14, 2007 in Würzburg, Germany.

Cancer Immunol Immunother 2008 Jan 6;57(1):135-42. Epub 2007 Oct 6.

Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333, ZA Leiden, The Netherlands.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00262-007-0408-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045121PMC
January 2008

Studies of S-adenosylhomocysteine-hydrolase polymorphism in a Croatian population.

J Hum Genet 2006 5;51(1):21-24. Epub 2005 Nov 5.

Institute of Human Genetics, Faculty of Medicine, University of Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany.

Recently, a proven case of human S-adenosylhomocysteine-hydrolase (SAHH) deficiency was reported in a Croatian boy. As molecular analysis of the SAHH gene in this case revealed two different mutant alleles, we investigated the polymorphism of human SAHH in a total of 237 red blood samples from unrelated Croats using starch gel electrophoresis and an enzyme-specific staining procedure. From the relative enzymatic activity of SAHH--determined by densitometric assessment of electrophoretic patterns, and calculated on the basis of the protein concentration of the red blood cells-we detected three individuals as being heterozygous for an SAHH 0-allele. Moreover, a total of four different electromorphic SAHHs have been observed, giving allele frequencies calculated as SAHH 1 = 0.941, SAHH 2 = 0.032, SAHH 3 = 0.006, SAHH 4 = 0.015, and SAHH 0 = 0.006.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10038-005-0315-zDOI Listing
March 2006